Artificial Light at Night Disrupts Circadian and Metabolic Gene Expression in the Green Anole Lizard (Anolis carolinensis): A Transcriptomic Analysis

21 Artificial light at night (ALAN) disrupts natural light-dark cycles, posing ecological challenges for wildlife 22 in urban areas. Here we investigated the effects of ALAN on gene expression in the brain, liver, and 23 skin of green anole lizards (Anolis carolinensis) whose urban populations are increasingly exposed to 24 light pollution. To identify genetic pathways impacted by ALAN exposure we analysed expression of 25 genes associated with circadian and metabolic regulation at midday, midnight and at midnight with 26 artificial light. Differential expression analysis revealed that clock-related genes (PER1, NR1D1, CRY2) 27 were significantly altered in the brain, liver, and skin following ALAN treatment and genes involved in 28 glucagon regulation ( GCG) and lipid metabolism ( NOCT) were differentially expressed in the liver, 29 indicating metabolic disruptions. Skin exhibited unique responses to ALAN suggesting that repair 30 responses may be altered as genes related to cellular processes , such as wound healing , were 31 upregulated under normal light and dark conditions. Our findings also show that ALAN disrupts core 32 circadian genes, impacting physiological processes including hormone regulation, glucose 33 homeostasis, and potentially reproductive cycles. This study provides the first transcriptomic evidence 34 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 2 of the effects of light pollution on green anoles, highlighting the need to preserve natural light cycles 35 in urban habitats. An interactive online database developed for this study allows further exploration 36 of gene expression changes, to promote research on artificial light-polluted environments. 37 38

Artificial light at night (ALAN), Circadian rhythm disruption, Green anole ( Anolis 39 carolinensis), Lizard, Metabolic regulation, Reptile, Transcriptomics 40 41

42 Light pollution, defined as the alteration of natural light -dark cycles by artificial light sources, has 43 become a persistent environmental concern worldwide, particularly in urban areas. The disruption of 44 a natural light -dark cycle can have detrimental effects on wildlife physiology and behaviour . These 45 disruptions are best-studied in mammals. For example, it has been shown that foraging behaviour in 46 the nocturnal Mongolian five-toed jerboa (Allactaga sibirica) is altered following exposure to artificial 47 light at night (ALAN; Zhang et al., 2020) and other nocturnal rodents such as wild mice (Mus musculus) 48 decreased their normal activity levels when artificial light was present (Oosthuizen et al., 2024). ALAN 49 has also been reported to have a negative effect on homeostasis in the spiny mouse ( Acomys 50 cahirinus) as chronic elevated cortisol levels and higher mortality rates were observed (Vardi-Naim et 51 al., 2022), and in laboratory mice ALAN prevented weight gain (Melendez-Fernandez et al., 2023). 52 Population size in wild mammalian species can be heavily impacted by urbanisation and light pollution 53 through effects on the reproductive clock . For example, in tammar wallab ies (Macropus eugenii), 54 ALAN suppressed the melatonin levels and delayed births (Robert et al., 2015). 55 ALAN has also been reported to affect other groups of wild vertebrates. Studies have shown 56 that artificial light not only affects bird behaviour , but also health and reproduction by altering 57 physiology and activity cycles (Amichai & Kronfeld -Schor 2019; Dominoni et al., 2013). Furthermore, 58 illumination at night can severely impact bird migration patterns, as well as avian perceptions of 59 habitat quality as illuminated areas are avoided (Adams et al., 2021). Studies in amphibians have 60 shown that artificial light exposure can change American toad ( Anaxyrus americanus) activity cycles 61 (Dananay & Bernard, 2018) with the potential to influence the ecosystem equilibrium as different 62 phenotypes can alter predator perception and amphibian population sizes (Shidemantle et al., 2022). 63 ALAN has also been shown to affect offspring behaviour in fish . Zebrafish (Danio rerio) that were 64 exposed to constant artificial light, not only showed altered behaviour , but F1 offspring born from 65 ALAN-exposed mothers displayed less frequent movement and shorter movement distances despite 66 never being exposed to ALAN themselves (Lim et al., 2024). 67 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 3 Reptiles are frequently exposed to light pollution in urban habitats, and the effects of artificial 68 light on their physiology and behaviour is less understood. The green anole lizard (Anolis carolinensis) 69 is a valuable species for studying the effects of ALAN as their behaviour and physiological processes, 70 such as circadian rhythms, thermoregulation, and reproduction , are strongly influenced by light, 71 including ALAN (Bayard 1974; Taylor et al., 2022). Green anoles are small, diurnal, arboreal lizards that 72 are commonly found in habitats ranging from dense forests to urban areas and are native to the south 73 eastern United States. Effects of ALAN in green anoles include increased nocturnal foraging and display 74 behaviour, reduced daytime activity, and changes in reproductive organ size (Taylor et al., 2022) and 75 such behavioural shifts are likely mediated by changes at the genetic level . Biological internal clocks 76 regulate daily cycles of physiological activity, a nd are controlled by complex genetic networks that 77 respond to external light cues. Disruption of these natural light-dark cycles by ALAN can interfere with 78 the circadian system through the expression of circadian rhythm -related genes to impact a range of 79 biological processes (Ouyang et al., 2018; Taylor et al., 2022; Thawley & Kolbe, 2020). Previous notable 80 work in reptiles has reported the effect of circadian rhythm disruption on metabolism and energy 81 regulation as ALAN has been shown to impact liver clock gene expression e.g., PER1 and GCG, leading 82 to metabolic imbalances, weight gain and glucose intolerance (Guan & Lazar 2022; Park et al., 2019). 83 Valuable insights into the underlying genetic and physiological impacts of altered light -dark 84 cycles by artificial light exposure on the disruption of core clock genes, including PER1, CRY1, NR1D1, 85 and BMAL1, has been extensively studied in laboratory mice (Bugge et al., 2012; Sato et al., 2004). 86 ALAN exposure in mice has been reported to affect the rhythmic gene expression of PER1 and NR1D1, 87 which play key roles in maintaining circadian stability. These genes act as transcriptional regulators 88 that link light exposure to physiological rhythms (Chauvet et al., 2016). Disruption by ALAN can cause 89 “phase shifts” in feeding, energy metabolism, and sleep -wake cycles, leading to desynchronization 90 between internal rhythms and the external environment. For example, nocturnal exposure to light 91 disrupts CRY2 and PER2 expression which results in altered sleep patterns, hormonal imbalances, and 92 alterations in glucose metabolism (Kalsbeek et al., 2010; Grunst et al., 2023). Other circadian -93 regulated genes, such as NOCT, are involved in lipid metabolism and NOCT expression is altered in 94 response to ALAN, which results in changes in lipid storage and transport (Kulshrestha et al., 2023). 95 Recent advances in transcriptomic analysis allow for a more detailed investigation into the 96 molecular effects of artificial light exposure in green anole lizards. Using the latest annotated green 97 anole genome (AnoCar2.0v2), gene expression profiling was used in this current study to identify 98 differentially expressed genes (DEGs) associated with artificial light exposure, highlighting potential 99 molecular mechanisms by which ALAN affects behaviour and physiology. Green anoles also exhibit 100 unique adaptations that make them suitable for studying photoreception beyond ocular tissues. In 101 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 4 addition to retinal opsins, anoles express photoreceptive proteins in extra-retinal tissues, such as the 102 skin and brain, potentially allowing them to detect environmental light changes directly through these 103 structures (Porter et al., 2011; Perez et al., 2019). These expression patterns suggest that there is a 104 complex sensory network that could contribute to light-dependent behaviour and physiology in urban 105 environments. 106 Given the ecological importance of lizards and the potential implications of light pollution for 107 health and behaviour, this study investigated the effects of ALAN on gene expression in the green 108 anole brain, liver, and skin . By analysing changes in gene expression across tissues, specific genetic 109 pathways regulating circadian rhythms and metabolic processes were found to be affected by ALAN. 110 Understanding these molecular responses provides a foundation for assessing the broader ecological 111 impacts of light pollution on vertebrates, as well as informing conservation strategies to mitigate the 112 effects of urbanisation on wildlife. 113 114

115 Animal capture and housing 116 Twenty-four free-living adult green anole lizards (Anolis carolinensis; twelve of each sexwere captured 117 in the breeding season, in May 2024, on the urban campus of Trinity University, San Antonio, Texas, 118 USA, during daylight hours. Green anole lizards were collected by using a dental floss loop attached to 119 an extendable fishing pole or by hand and were transported individually to the Trinity University 120 vivarium in cotton bags. On the day of capture, the body mass of each anole was measured to the 121 nearest 0.1 g using a Pesola spring scale and snout -vent length measured to the nearest mm using a 122 clear plastic ruler (Males: range 52-69mm, average 62mm; Females: range 51-57mm, average 55mm). 123 Each individual was given a unique identification number on the lower jaw using a non -toxic 124 permanent marker. Each anole was then randomly assigned to one of three treatment groups: 125 Midday, Midnight, or ALAN. Sex was determined by the presence of a dewlap; four anoles of each sex 126 were assigned to each treatment group. 127 All green anoles were housed in the Trinity University vivarium following standard anole care 128 procedures for a minimum of four days prior to tissue collection (Sanger et al. , 2008). Pairs of anoles 129 (one of each sex) were assigned to the same treatment and housed together in large Kritter Keeper™ 130 cages (37.5 x 21.0 x 28.0 cm 3; Lee’s Aquarium and Pet Products, San Marcos, CA, USA). Cages 131 contained Zilla Green Terrarium Liner ™ (Zilla, Franklin, WI, USA), 2 PVC pipe perches, a wire mesh 132 hammock, and a nest box where females could lay eggs (i.e., a plastic flower pot with moist sphagnum 133 moss). Cages were misted with water daily to provide drinking water, and each anole was fed 134 approximately every other day between 12:00 and 18:00. At each feeding, two or three crickets were 135 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 5 dusted with Zoo Med Repti Calcium ™ supplement (Zoo Med Laboratories, Inc., San Luis Obispo, CA, 136 USA). 137 In the vivarium, green anoles were housed together in one room for the Midday and Midnight 138 treatments, and housed in an adjacent room for the ALAN treatment. The humidity and temperature 139 ranged from 51 – 62 %, and 25.1 - 28.1 oC respectively, with similar conditions in both rooms. All cages 140 were kept under standard lighting conditions on a 12.5 light: 11.5 dark cycle (room ceiling lights on at 141 06:00) to mimic the natural light -dark cycle for the month of May in San Antonio, Texas, USA. Two 142 Reptisun 5.0 UVB light bulbs (Zoo Med Laboratories, Inc., San Luis Obispo, CA, USA; emission peaks at 143 410, 440, 550 and 580 nm and broadband emission centred at 350 nm) were positioned over each 144 cage to simulate the full spectrum of natural sunlight. To mimic daily dawn and dusk, room ceiling 145 lights (32-watt GE T8 Starcoat ECO bulbs, GE, Boston, MA, USA; emission peak 450 nm and broadband 146 emission centred at 600 nm) were switched on 30 min. before and 30 min. after the cage lights. Ceiling 147 lights were switched off at 19:30 in both rooms, but in the ALAN treatment room, a street lamp (D802-148 LED 12 ʹʹ low -profile area light; Deco Lighting, Inc. Commerce, CA, USA) identical to those used for 149 nocturnal lighting on Trinity University’s campus, was switched on. The street lamp was covered with 150 black mesh deer cloth to provide a light intensity of 1.21 µmol / m / s (approx. 89.6 lux; SD = 0.14 µmol 151 / m2 / s;) at a distance of 180 cm from the lizard cages in the ALAN room (see Taylor et al., 2022). This 152 light intensity mimics the light intensity of nocturnal lighting on campus (1.33 µmol/m2; SD = 0.16 153 µmol /m2 / s (approx. 98.5 lux / s; Taylor et al., 2022). The streetlamp was switched off at 06:00, when 154 the ceiling lights of the room were switched on. ALAN green anoles were maintained in this street light 155 treatment for 3-5 days prior to cull and tissue collection. 156 Tissue collection 157 Green anoles were rapidly decapitated without prior anaesthesia to avoid any confounding 158 anaesthetic effects on RNA expression. Brain (containing the pineal gland), eyes, dorsal skin, ventral 159 skin, liver, and testes or ovaries were collected in under 7 min. 33 s and flash frozen on dry ice and 160 stored at -80 oC until they were shipped to BGI Genomics (San Jose, CA, USA). Midday treatment 161 dissections were performed between 12:48 to 13:45, and Midnight and ALAN treatment from 22:30 162 to 00:42. Decapitation for the Midnight treatment group was performed in the dark under red torch 163 light (HQRP, Harrison, NJ; emission peak in the red spectrum at 650 nm). To control for this additional 164 illumination, ALAN treatment lizards were also illuminated by the same red torch light during 165 decapitation. 166 Gene expression analysis 167 Tissues were shipped on dry ice to BGI Genomics (San Jose, CA, USA) where they were processed for 168 RNA extraction, library preparation and sequencing. The quality of the RNA was checked before 169 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 6 proceeding to library preparation to ensure a RIN of at least 7.0. Samples that did not pass the filter 170 were discarded from the analysis. Raw fastq files were processed by trimming using trimmomatic 171 (http://www.usadellab.org/cms/?page=trimmomatic) with the following parameters: 172 ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10; SLIDINGWINDOW:10:30; LEADING:28; TRAILING:28; 173 MINLEN:75. After trimming the quality of the reads was checked using FASTQC (https://github.com/s-174 andrews/FastQC). Once the parameters were checked, reads were aligned using STAR aligner version 175 2.7.11a (https://github.com/alexdobin/STAR), with the ENSEMBL green anole ( Anolis carolinensis ) 176 genome and gtf (AnoCar2.0v2, Anolis_carolinensis.AnoCar2.0v2.112.gtf) as references. BAM files 177 were counted using featurecounts (https://subread.sourceforge.net/). 178 Downstream statistical analysis was performed on R version 4.4.0. using the package Deseq2 179 (Love, et. al., 2014 ). Volcano, PCA, heatmaps and boxplots were drawn using ggplot2 180 (https://ggplot2.tidyverse.org/), and GO enrichment analysis was performed using the package 181 genekitr (Liu, et.al.,2023) Venn diagrams were generated using the library VennDiagram (https://r -182 graph-gallery.com/14-venn-diagramm). GO classification analysis was performed in Panther 183 (https://www.pantherdb.org/), by feeding the ENSEMBL IDs and selecting the green anole genome 184 (Anolis carolinensis ) as a reference. The web App was developed using R shiny 185 (https://shiny.posit.co/), using Plotly to make any interactive plot interactive ( https://plotly.com/r/). 186 Genes were considered differentially expressed if they showed a log fold change of more than or equal 187 to 1 or less than or equal to -1 and a p adjusted value of less or equal to 0.05. 188 Data availability 189 Raw data can be found in: GEOXXXX. And any datasets used during the current study are available 190 from the corresponding author upon request. 191 192

193 This study provides, to our knowledge, the first transcriptomic analysis of a reptile species in response 194 to ALAN exposure. As an initial approach, a PCA analysis was conducted to examine variability among 195 groups by comparing samples from lizards collected at Midday, Midnight (no light), and exposed to 196 ALAN (midnight with artificial light ). From this analysis, three distinct clusters emerged: one 197 comprising of liver, a second and larger cluster containing skin (both dorsal and ventral), testes, and 198 ovary samples with a subtle separation between skin and gonad groups, and a third cluster formed by 199 brain and eye samples (Figure 1). A cluster composed of the skin, testes, and ovary samples showed 200 minimal differences between light conditions. ALAN and Midnight liver samples clustered closely, 201 while samples collected during Midday appeared more dispersed. For the brain, a clustering pattern 202 emerged between Midday and Midnight samples, regardless of light exposure, although some outliers 203 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 7 could be identified. No clustering was observed for liver or skin within light treatments (see Appendix 204 1). 205 To further explore the effects of ALAN on different green anole tissues, ALAN samples were 206 compared to those taken during the Midday and those taken at Midnight, to identify genes 207 differentially expressed under “normal” day -night cycles and those differentially expressed under 208 ALAN. Analysis was specifically focused on brain, which regulates circadian rhythms (Benca et al., 209 2009); liver, which reflects metabolic responses to artificial light (Thawley et al., 2020) and skin, which 210 our unpublished data suggests may exhibit high photoreceptor variability (Trejo-Reveles et al., 211 submitted). By comparing Midnight samples to those exposed to ALAN, circadian rhythm -related 212 genes were found to be differentially expressed across tissues. Specifically, in the brain, the clock gene 213 PER1 (up-regulated; ) and clock protein regulator NR1D1 (down-regulated) emerged as the top 214 differentially expressed genes. In the liver, genes associated with glucagon synthesis, such as GCG (up-215 regulated) and GOT1L1 (down-regulated) , were differentially expressed along with PER1 (up-216 regulated) . In addition, circadian-related genes, including DBP (down-regulated), and NR1D1 (down-217 regulated), were also differentially expressed in the skin. In the skin, a similar pattern to that observed 218 in the brain and liver was identified, but additional genes, such as CEBPD and M13A, were also 219 differentially expressed (Figure 2). When these results were compared to those obtained when 220 samples were subjected to a natural light -dark exposure, PER1 remained differentially expressed, 221 particularly in the liver. Other genes, such as NOCT, KLF9, and PPR31B, also showed differential 222 expression in this natural light comparison. Similar results were observed for the brain, where the 223 majority of the genes that were DE in the Midday vs ALAN comparison were also DE in the Midday vs 224 Midnight comparison. In the skin, a significantly lower number of genes were found DE when the 225 tissue was not exposed to ALAN (Figure 2). 226 Differences between ALAN exposure and natural light schedule 227 To further explore genes differentially expressed under a normal light -dark cycle compared to those 228 under ALAN exposure, a Venn diagram analysis followed by gene ontology (GO) classification was 229 conducted. The results obtained in the pairwise comparisons between Midday and Midnight and 230 Midday and ALAN were compared and focused on both the shared DEGs and the DEGs that were 231 exclusive to ALAN exposure. In the brain, only 26% of the DE genes were exclusive to the Midday vs. 232 ALAN comparison (Figure 3). GO classification indicated that most of these genes were related to 233 cellular process categories, including cell division and adhesion (see Appendix 2). Notably, CRY2, a 234 circadian rhythm modulator was exclusively differentially expressed under ALAN exposure. 235 In the liver, only 12.8% of DEG’s were shared between light-exposed and dark samples (Figure 236 3). Similar to the brain, shared terms in the liver were mostly related to cellular process, such as cell 237 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 8 death and division. Some circadian -clock genes, including PER1, CRY1, and PER2, were commonly 238 expressed. Genes exclusively DE in the liver under ALAN were primarily involved in metabolic 239 processes, such as glycolysis. Interestingly, the F -box domain -containing protein, associated with 240 circadian rhythm regulation, was DE in the Midday vs. ALAN comparison. NOCT (aka Ccrn4l), which is 241 involved in the liver circadian clock and lipid metabolism (Kulshrestha et al., 2023), was downregulated 242 under natural light-dark cycles but absent in DEG lists obtained under ALAN (Figure 3). 243 The largest discrepancy in DE genes was observed in the skin when comparing ALAN exposure 244 to the other treatment groups. Only 3% of DEG’s were shared across comparisons, while a striking 245 78% were exclusive to ALAN exposure. Similar to the results observed for brain and liver, circadian 246 clock-related genes, such as CRY1, CRY2, and PER1, were among the 56 shared genes. In the DE list 247 exclusive to ALAN exposure, cellular process, localization, and pigmentation were identified as primary 248 categories, with no circadian rhythm-related terms appearing exclusively enriched in the Midday vs. 249 ALAN comparison (Figure 3). When comparing dorsal and ventral skin (data not shown), we observed 250 a differential expression of OPN5, a non -visual opsin previously detected in the skin of green anoles 251 (Trejo-Reveles, et al., submitted). Notably, OPN5 expression was differentially regulated regardless of 252 skin exposure to artificial light at night (ALAN). Interestingly, the dorsal skin exhibited the highest 253 levels of OPN5 expression (Appendix 3), surpassing even the expression levels found in brain tissue . 254 There were no sex differences in gene expression patterns with most of the genes following the same 255 expression patterns regardless of the tissue type. Key genes for each comparison are summarized in 256 Table 1, and their respective expression patterns are found in Appendix 3 . GO analysis showed 257 significant enrichment only in the brain, where both Midday vs. ALAN and Midday vs. Midnight 258 comparisons highlighted the regulation of circadian rhythms. No terms were significantly enriched in 259 the skin or the liver (p-value ≤ 0.05). 260 When comparing the Night vs. ALAN conditions, we observed the highest number of 261 differentially expressed genes (DEGs) in the skin, exceeding the numbers identified in the previous 262 two comparisons (2023). Notably, GRIA2 was upregulated at levels similar to those observed in the 263 ALAN vs. Midday comparison, with additional genes such as GRM5 (up regulated) also showing 264 differential expression. New DEGs identified included NPY, SNAP25, and DNER, all of which were 265 upregulated, while genes such as CCDC were downregulated (Appendix 4 A). 266 In the brain, the ALAN vs. Midnight comparison revealed only 50 DEGs, representing the 267 lowest number among all comparisons. Notably, no genes were downregulated in this condition. 268 Among the upregulated genes, the Growth Hormone-Releasing Hormone Receptor (GHRHR) was the 269 annotated gene that exhibited the most striking upregulation (p -value of 7.93e-06, log fold change 270 2.93). Novel transcripts, including ENSACAG00000006458, ENSACAG00000011927, and 271 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 9 ENSACAG00000017407 ( this last one presumed to encode for MYL4), were also up-regulated. Other 272 DEGs included circadian -related genes such as PER1 and interestingly FOS was also up regulated in 273 this comparison. (Appendix 4B). 274 Liver tissue displayed a total of 259 DEGs, the fewest observed across the comparisons. While 275 some genes were consistent with those identified in the Midday vs. ALAN comparison, such as CGC 276 and MT-ND6, novel targets also emerged. Among the newly identified genes, GAD2 was upregulated, 277 whereas ACTA1 and KLF9 were downregulated. (Appendix 4C). 278 Further exploration of the dataset 279 This comprehensive study provides valuable insights into the genetic impacts of artificial light 280 exposure on green anoles, highlighting significant changes in circadian rhythm and metabolic gene 281 expression across tissues. Due to the scope and complexity of the data, it is challenging to capture and 282 summarise all possible comparisons and interactions within a single report. To address this, we 283 developed a publicly accessible online database to allow researchers to further explore the dataset in 284 depth, analyse specific genes, and examine differential expression patterns tailored to additional 285 research questions. This interactive resource is available at 286 https://vtrejor.shinyapps.io/green_anole_rnaseq/. It provides a user-friendly platform for visualising 287 and downloading data, supporting a more nuanced understanding of how light exposure affects 288 genetic expression in reptiles. By making this resource available, we hope to facilitate further research, 289 collaboration, and discovery in the field of vertebrate biology and conservation. 290 291

292 This study presents the first transcriptomics database focusing on ALAN effects in lizards, which given 293 their distribution in urban habitats (French et al. 2018) , are frequently subjected to light pollution 294 (e.g., Thawley and Kolbe 2020; Taylor et al., 2022). This report focused on the effect of ALAN on 295 circadian and metabolic responses in the brain and liver as well as opsin expression in the skin. To 296 complement these findings a comprehensive publicly accessible database was developed for 297 researchers to further explore the effects of ALAN. 298 In the brain, which included the pineal gland, similar patterns of DEGs under ALAN versus 299 natural light -dark cycles were observed, which is consistent with the brain containing the master 300 internal biological clock (Miller et al., 2015). However, some genes displayed a significant difference 301 in log-fold changes, or were not differentially expressed at all in one of the comparisons. For example, 302 NR1D2 was upregulated when Midday vs. Midnight was compared; its ortholog, NR1D1, was 303 downregulated in the same comparison. Under ALAN exposure, only NR1D1 was differentially 304 expressed. Both NR1D1 and NR1D2 are associated with photoperiodism, as they play a key role in the 305 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 10 regulation of circadian rhythms by modulating gene expression in response to light. NR1D1 (also 306 known as REV -ERBα) and NR1D2 (REV-ERBβ) act as transcriptional repressors within the molecular 307 circadian clock, influencing the stability and amplitude of circadian rhythms through the repression of 308 genes involved in metabolic, inflammatory, and behavioural pathways (Preitner et al., 2002; Bugge et 309 al., 2012). These genes are particularly sensitive to light, which entrains their rhythmic expression 310 patterns, providing a direct link from external light-dark cycles to internal biological rhythms (Sato et 311 al., 2004). Although the role of these genes in the regulation of the reptile circadian rhythm is less 312 understood, studies in other vertebrates suggest that these genes are crucial for photoperiodic 313 responses, including those associated with feeding and seasonal reproduction (Chauvet et al., 2016). 314 Related to light detection, one interesting finding was that RH2, a green light-sensitive opsin, showed 315 differential expression in the brain when exposed to ALAN. While RH2 is typically studied in the eye, 316 prior data links its involvement to seasonal changes in circulating testosterone in male green-spotted 317 grass lizard Takydromus viridipunctatus (Tseng et al., 20 18. Unpublished data from our laboratory 318 (Trejo-Reveles et al., submitted) suggest that opsins, particularly OPN5, vary in expression across 319 green anole tissues. The role of opsins in response to ALAN is yet to be investigated, but this study 320 highlights the importance of extra retinal photoreceptors in structures such as the brain, pineal gland 321 and skin. 322 In the liver, GCG, which encodes glucagon, was differentially expressed under ALAN, 323 suggesting that disrupted circadian cycles may alter hormone production in lizards (Martin & White, 324 2016). Glucagon plays a crucial role in maintaining glucose homeostasis by stimulating glycogen 325 breakdown and glucose release, particularly during fasting states. Its secretion follows a circadian 326 rhythm regulated by the liver’s internal clock and feedback mechanisms from other organs, such as 327 the pancreas and the hypothalamus, aligning glucagon levels with the body’s metabolic needs over 328 the day-night cycle (Kalsbeek et al., 2010; Vieira et al., 2015). The rhythmic release of glucagon is 329 driven, in part, by core clock genes, including PER1, and is influenced by light exposure, which can 330 disrupt glucagon’s circadian oscillations, leading to altered glucose metabolism and potential 331 metabolic imbalance (Grunst et al., 2023). This regulation is essential for energy balance, as glucagon 332 levels peak during nocturnal phases, preparing the body for fasting periods, and are suppressed during 333 feeding periods (Guan & Lazar, 2022). PER1 was differentially expressed under both conditions, 334 indicating that altered light-dark cycles could affect clock gene expression in the liver, potentially as a 335

of an imbalanced glucagon regulation (Ando et al., 2013). In addition, NOCT, which is involved 336 in the liver circadian clock and lipid metabolism (Kulshrestha et al., 2023), was downregulated under 337 natural light -dark cycles. Knockout studies in mice have demonstrated NOCT’s roles in cellular 338 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 11 differentiation and metabolism, with implications for higher energy expenditure, and reduced 339 adiposity (Le et al., 2019; Abshire, et al., 2020). 340 In the skin, many genes were upregulated under ALAN exposure. Notable genes include 341 GRIA2, which is associated with circadian rhythm maintenance in the mouse brain, and GRM5, which 342 is involved in sleep-wake cycles (Raap et al., 2015; Taylor et al., 2022). Genes that were differentially 343 expressed in normal light-dark cycles in the skin were generally not associated with circadian rhythms, 344 except for NR1D1. Instead, genes such as PNK4, involved in wound healing, and CEBPD, related to cell 345 death and cell proliferation (Balamurugan & Sterneck 2013) were differentially expressed. Our 346 previous unpublished data (Trejo -Reveles et al., submitted) have shown that OPN5 expression is 347 downregulated in ventral skin exposed to light compared to dorsal skin. These data presented in this 348 study confirm that OPN5 is down-regulated in ventral skin regardless of ALAN exposure. This suggests 349 a putative role of extra retinal photoreceptors in the green anole and highlights the importance of 350 natural light-dark cycles. 351 Several reptilian studies have focused on the effects of artificial light exposure on behaviour, 352 but have not specifically identified genetic markers to indicate stress or triggers for those behavioural 353 changes. Lizards, which are common in the wild including urban environments, face significant 354 challenges from light pollution. For example, previous studies using a diversity of lizard species have 355 shown that nocturnal activity and foraging levels are increased under ALAN , but this is generally 356 associated with reduced performance during the day (e.g., Martin et al. 2018, Mauer et al. 2019, Oda 357 et al. 2020, Kolbe et al. 2021). Another study showed that green anoles exposed to ALAN were more 358 active at night, using the nocturnal artificial light to explore, forage, and display. During the day, ALAN 359 exposed lizards exhibited reduced activity, and displayed increased fat pad and testes sizes, suggesting 360 shifts in metabolic and reproductive processes (Taylor, et al., 2022). Our findings align with these 361 observations; for instance, genes such as GRM5, associated with nocturnal activity, were upregulated 362 under ALAN exposure. ALAN has been shown to have no effect on green anole offspring quality (Clark 363 et al., 2017), however clear ALAN induced changes in adult lizard ovaries was observed as TTR was 364 upregulated in this study (data not shown). TTR accumulates in the choroid plexus during the dark 365 phase of the circadian rhythm, and it is known to be influenced by sex, age and circadian rhythms. In 366 mice, TTR plays a role in preparing the uterus for embryo implantation potentially influencing offspring 367 quality (Fame et al., 2023; Duarte et al., 2020; Diao et al., 2010). 368 In conclusion, t his study provides the first transcriptomic analysis to examine the effects of 369 ALAN on a reptile species . In addition, these data provide a valuable public resource for researchers 370 interested in the effects of light pollution on reptiles. The findings reveal that ALAN exposure disrupts 371 the expression of key circadian and metabolic genes across tissues, highlighting the sensitivity of these 372 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 12 lizards to light pollution. Specifically, the differential expression of clock -related genes such as PER1, 373 NR1D1, and CRY2, alongside glucagon-related genes in the liver, underscores the influence of artificial 374 light on fundamental physiological processes, from circadian rhythm regulation to glucose 375 homeostasis. The observed modulation of photoreceptor genes, particularly in skin and brain, 376 provides evidence of extra -retinal photoreception in lizards and suggests an adaptive response that 377 may be crucial for these reptiles to cope with light -polluted environments. While these findings 378 provide a comprehensive understanding of how ALAN exposure affects the gene expression profile in 379 green anoles, they also emphasise the need to maintain natural light-dark cycles in wild urban habitats 380 to support optimal physiological functioning. Investigating the functional roles of genes such as OPN5 381 and RH2 in extra-retinal and retinal photoreception, and their potential contribution to behavioural 382 and physiological adaptations in light -polluted environments, will deepen our understanding of 383 photoreception beyond ocular tissues. Furthermore, comparative studies across reptile species 384 exposed to ALAN could reveal evolutionary adaptations to light pollution, offering insights into the 385 resilience of reptile populations in urbanised landscapes. Longitudinal studies tracking gene 386 expression changes across life stages will elucidate whether prolonged exposure to ALAN induces 387 cumulative genetic or phenotypic changes, particularly concerning reproductive fitness and stress 388 responses. Future studies will embrace a transcriptomic approach to further investigate the broader 389 impacts of ALAN on reptilian biology. Indeed, ATACseq, and single cell sequencing, will be key to the 390 understanding which cell populations are important for physiological and behavioural adaptation in 391 our rapidly illuminated world. These findings will be instrumental for conservation efforts aimed at 392 mitigating light pollution and preserving natural light cycles, which are integral to the health and 393 survival of animals in their natural habitats. 394 395

396 This work was supported by an International Institutional Award to the University of Edinburgh 397 (BB/Y51410X/1) and Roslin Institute Strategic Grant (BBS/E/RL/230001C) funding from the UK 398 Biotechnology and Biological Sciences Research Council to Simone L. Meddle along with financial 399 support from the Trinity University Office of Academic Affairs to Michele A. Johnson. We would like 400 to thank Dale Cochran and members of the Johnson Lab for all of their fantastic help in the laboratory 401 and field. Green anole collection was performed under Scientific Permit SPR -0310-045 to Michele A. 402 Johnson from Texas Parks and Wildlife Department, with approval from Trinity University’s Animal 403 Research Committee, protocol 051122-MJ2. 404 405 Author contributions 406 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 13 V.T-R, M.A.J, and S.L.M. designed the research; V.T -R, M.A.J, G.E.A , F.J.P and A.R.J. performed the 407 research and V.T-R, M.A.J, and A.R.J. analysed the data; V.T-R, M.A.J, and S.L.M. wrote the manuscript. 408 All the authors were involved in drafting and revising the manuscript. 409 410 Competing interests 411 The other authors have no conflict of interest. 412 413 Table 1. 414 Tissue DE key genes Possible implications Brain PER1, NR1D1, NR1D2, CRY1, CRY2, RH2, FOS, GHRHR, ENSACAG00000017407 (MYL4) Disruption of circadian regulation and potential effects on hormonal rhythms. NR1D1/NR1D2 and CRY2/CRY1 involvement in photoperiodism may affect seasonal and daily physiological processes, while RH2 expression suggests sensitivity to green light, possibly influencing testosterone levels and behavior. FOS upregulation may indicate stress or immediate early gene activation. GHRHR upregulation suggests impacts on growth hormone signaling. The novel transcripts, including MYL4, could imply emerging regulatory pathways influenced by ALAN. Liver GCG, PER1, NOCT, F-box domain-containing protein, GAD2, ACTA1, KLF9,MT-ND6 Altered glucagon production and glucose homeostasis due to GCG and PER1 regulation, impacting metabolism and energy balance. NOCT downregulation suggests disruption in lipid metabolism and resistance to obesity, potentially leading to metabolic dysregulation. GAD2 upregulation implies involvement in neurotransmitter signaling or energy metabolism, while ACTA1 and KLF9 downregulation highlight .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 14 structural and transcriptional regulation disruptions under ALAN exposure. Skin GRIA2, GRM5, OPN5, PND4, NPY, SNAP25, DNER, CCDC Increased expression of GRIA2 and GRM5 could influence circadian rhythm maintenance and sleep-wake cycles. The downregulation of OPN5 in ventral skin indicates potential roles for extra-retinal photoreceptors in light perception. PND4 involvement in wound healing and cell proliferation suggests additional responses in the skin under ALAN. The upregulation of NPY, SNAP25, and DNER may reflect roles in non-neuronal processes such as tissue remodeling, signaling pathways, or cellular interactions in the skin. CCDC downregulation highlights gene-specific suppression under ALAN 415 Figure and Table Legends 416 Table 1. 417 Key genes found differentially expressed (DE) in each tissue and the possible connection to artificial 418 light at night (ALAN) in green anole lizards. 419 420 Figure 1. 421 PCA plot comprising all samples from different tissues along with Midday, Midnight and ALAN 422 treatments in green anole lizards. Shape depicts tissue, whilst colour illustrates treatment. 423 424 Figure 2. 425 Volcano plots of differentially expressed genes (DEG). A - C: genes differentially expressed when 426 comparing Midday vs ALAN; D - F: genes differentially expressed when comparing Midday vs Midnight. 427 Comparisons are shown in the following order: Brain, Liver and Skin. Yellow dots depict up-regulated 428 genes whilst blue dots represent down -regulated genes; black dots are non -significant genes. 429 Significant genes were chosen based on Log fold change ( = 1) and p adjusted value (</= 430 0.05). 431 432 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 15 Figure 3. 433 Venn diagrams of DEG for Midday vs ALAN or Midday vs Midnight comparisons. Each of the Venn 434 diagrams show the similarity between comparisons for A: brain B: liver and C: skin. 435 436 Figure 4. 437 Enriched GO terms when comparing brain issue Midday vs ALAN. The colour is based on the p 438 adjustment of the enrichment. Only two categories showed significant enrichment: rhythmic process 439 and circadian rhythm. 440 441 Appendix Figure Legends 442 Appendix 1. 443 PCA plots of individual tissues from green anoles; the colour depicts the treatment and the shape 444 depicts the sex. None of the PCA plots show clear clustering between different light ing conditions or 445 sex. 446 447 Appendix 2. 448 GO categorization of DEG that were exclusively found when comparing Midday vs ALAN, exclusively 449 found when comparing Midday vs Midnight, and terms that were found in both comparisons (depicted 450 as common terms). Each colour represents a GO category. X axis is the category to which the genes 451 belonged to (common, Midday vs Midnight or Midday vs ALAN). Y axis is the percentage (1=100%). A. 452 Brain; B. Liver C. Skin. 453 454 Appendix 3. 455 FPKM values of genes of interest in different tissues, conditions and sex. Each boxplot represents a 456 light exposure condition. X axis represents the tissue; Y axis depicts the FPKM and colours represent 457 the lighting condition. Female and male individuals’ values are shown next to each other. 458 459 Appendix 4 460 ALAN vs Midnight comparison volcano plots 461 462 463 464 465 466 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 16

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It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 21 607 Figure 3 608 609 Figure 4 610 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 22 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627 628 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 23 Appendix 629 Appendix 1. 630 631 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 24 632 Appendix 2 633 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 25 634 Appendix 3 635 636 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 26 637 638 639 640 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 27 641 642 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 28 643 644 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 29 645 646 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 30 647 648 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint 31 649 Appendix 4 650 651 .CC-BY-NC 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprintthis version posted March 21, 2025. ; https://doi.org/10.1101/2025.03.20.644270doi: bioRxiv preprint